Remedy for infections

ABSTRACT

According to the present invention, there is provided a remedy for infections containing 15K granulysin as an active ingredient, which has no perceived side effect, and is effective.

TECHNICAL FIELD

The present invention is an invention relating to an infection remedyfor treating infectious diseases such as tuberculosis.

BACKGROUND OF THE INVENTION

In medical care, it goes without saying that the existence of a remedyfor infectious diseases is indispensable. At present, a number ofremedies for infections such as antibiotics and synthetic antibacterialagents are provided, and are used in medical care.

However, currently, an antibacterial agent which is mainly provided as aremedy for infectious diseases is in fact accompanied with anunavoidable problem of appearance of drug resistant bacteria. That is,sarcastic state continues in which by provision of a new antibacterialagent, a new drug resistant bacterium is produced.

For example, in tuberculosis occupying one of the highest number ofpatient with in single infectious disease, a tendency of recent increasebecomes a worldwide problem. Further, the existence of a drug resistantbacterium having resistance to almost all antibiotics has beenconfirmed, and this problem is being revealed.

Recently, it has been revealed that a molecule called granulysinexpressed in a Natural Killer cells (NK cell) or Cytotoxic T Lymphocytes

(CTL) has direct ability to kill and injure bacteria such asMycobacterium tuberculosis [Stenger, S. et al., Science 282,121-125(1998)].

Granulysin is produced as a precursor of 15K and, thereafter, processedinto 9K in a cytotoxic granule, and it is known that this 9K granulysinhas antibacterial activity (Pena, S. V. et al., J. Immunol, 158,2680-2688 (1997)). Further, a pathway is reported in which perforinwhich is the molecule derived from the same cytotoxic intragranuleperforates a target cell, granulysin enters into a cell therethrough,and kills and injures an infecting bacterium in a cell [Stenger, S. et.al., Science 282, 121-125 (1998)].

Like this, it is thought that granulysin plays an important role indefense for infection. It is hardly thought that active type 9Kgranulysin makes a bacterium thereto acquire resistance, and applicationto a remedy for infectious diseases having different characteristic froman antibiotic is contemplated. In addition, recently, the existence ofperforin pathway regarding removal of an intracellular infectiousbacterium by 9K granulysin is suspected (David, H. C. et al., J.Immunol, 167, 2734-2742 (2001)). Moreover, cytotoxity is perceived inthis molecule (Pena, S. V. et al., J. Immunol, 158, 2680-2688 (1997))and, when this is administered as such, considerable side effecttoxicity is feared.

A problem to be solved by the invention is to study granulysin infurther detail, and provide means for using this as a remedy forinfectious diseases.

DISCLOSURE OF THE INVENTION

In order to solve this problem, the present inventors intensivelystudied. As a result, the present inventors found out a novel pathwaywherein 15K granulysin is incorporated into a macrophage and,thereafter, activated to kill and injure bacteria phagocytosed into amacrophage.

That is, the present inventors found out that use of 15K granulysinshowing no cytotoxicity in itself as an active ingredient makes itpossible to provide an efficacious remedy for infections which haslittle side effect and to which bacteria can acquire little tolerance.

The present invention is the invention providing an infection remedy ofwhich active ingredient is 15K granulysin (hereinafter also referred toas the present remedy).

In the present invention, 15K granulysin is a protein, having amolecular weight of 15,000 (15k), consisting of 145 amino acids which islocalized in cytotoxic granule as described above. In a cytotoxicgranule, a part of 15K granulysin is cut, and 15K granulysin is presentas a protein having a molecular weight of 9,000 (9k) (Pena, S. V., etal., J. Immunol., 158, 2680-2688 (1997), Stenger, S., et al., Science,282, 121-125 (1998)).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a view showing results of study on antibacterial effect of 15Kgranulysin on Mycobacterium tuberculosis.

DETAILED DESCRIPTION OF THE INVENTION

Embodiments of the present invention will be explained below.

A. Active Ingredient of Present Remedy

15K granulysin used as an active ingredient of the present remedy can is5 be used by separating from a living body, but since 15K granulysin isa trace component in a living body, it is preferable to use as arecombinant protein obtained by expressing a gene encoding 15Kgranulysin.

Alternatively, it is also a suitable means to produce 15K granulysin ina living body, utilizing an intracorporeal expression vector for 15Kgranulysin in which a gene encoding 15K granulysin is inserted, as anactive ingredient.

(i) Recombinant Protein of 15K Granulysin

A sequence of a gene encoding 15K granulysin has been already reported(Jongstra, et al., J. Exp. Med, 165, 601), specifically, a gene having anucleotide sequence represented by SEQ ID NO: 1 and a 15K granulysinprotein having an amino acid sequence corresponding thereto. Based onthis, a gene encoding 15K granulysin is effectively prepared, and thisgene is expressed, whereby, a recombinant protein of 15K granulysin canbe prepared.

Specifically, a gene amplification product of a gene encoding 15Kgranulysin is prepared using nucleotide chains complementary to bothends of a sequence of a gene encoding 15K granulysin as an amplificationprimer, by a gene amplification method such as a PCR method.

This is transduced into a suitable gene expression vector, and desired15K granulysin can be obtained from a suitable host such as Escherichiacoli, Bacillus subtilis, yeast and insect cell transformed with such therecombinant vector.

It is preferable that, as a gene expression vector used herein, a vectorusually harboring a promoter and an enhancer at a region upstream of agene to be expressed, and a transcription termination sequence at aregion downstream of the gene is used.

In addition, expression of a 15K granulysin gene is not limited to adirect expression system. For example, a fused protein expression systemutilizing a D-galactosidase gene, a glutathione-S-transferase gene or athioredoxin gene may be used.

As a gene expression vector using Escherichia coli as a host, there areexemplified pQE, pGEX, pT7-7, pMAL, pTrxFus, pET, and pNT26CII. As avector using Bacillus subtilis as a host, there are exemplified pPL608,pNC3, pSM23, and pKH80.

In addition, as a vector using yeast as a host, there are exemplifiedpGT5, pDB248X, pART1, pREP1, YEp13, YRp7, and YCp50.

In addition, as a vector using a mammal cell or an insect cell as ahost, there are exemplified p91023, pCDM8, pcDL.SRa296, pBCMGSNeo,pSV2dhfr, pSVdhfr, pAc373, pAcYM1, pRc/CMV, pREP4, and pcDNAI.

These gene expression vectors can be selected depending on the purposeof expression of 15K granulysin. For example, when 15K granulysin isexpressed, it is preferable to select a gene expression vector for whichEscherichia coli, Bacillus subtilis or yeast can be selected as a host.When 15K granulysin is expressed even at a small amount so that it hasassuredly activity, it is preferable to select a gene expression vectorfor which a mammal cell or an insect cell can be selected as a host.

In addition, it is possible to select the existing gene expressionvector as described above, but a gene expression vector is appropriatelyproduced depending on a purpose, and this may be of course used.

Transfection of the gene expression vector in which a 15K granulysingene is inserted, into a host cell, and a transformation methodtherewith can be performed by the general method, for example, a calciumchloride method and an electroporation method in the case of Escherichiacoli and Bacillus subtilis as a host cell, and by the means such as acalcium phosphate method, an electroporation method and a liposomemethod in the case of a mammal cell and an insect cell as a host.

By the culture of the thus obtained transformant according to theconventional method, desired 15K granulysin is accumulated.

Medium used in such the culture can be appropriately selected dependingon a host. For example, in the case of Escherichia coli as a host, LBmedium and TB medium can be appropriately used and, in the case ofmammal cells as a host, RPMI1640 medium can be appropriately used.

Isolation and purification of 15K granulysin from a culture obtained bythis culture can be performed according to the conventional method. Forexample, isolation and purification can be performed by subjecting tothe culture to various treating operations utilizing physical and/orchemical nature of 15K granulysin.

Specifically, treatment with a protein precipitating agent,ultrafiltration, gel filtration, high performance liquid chromatography,centrifugation, electrophoresis, affinity chromatography using aspecific antibody, and dialysis method can be used alone or by combiningthese methods.

As described above, 15K granulysin can be separated and purified.

By using 15K granulysin as an active ingredient in a remedy forinfectious diseases, it is incorporated into a macrophage in blood, andactivated therein, whereby, it kills and injures bacteria, viruses, andfungi inducing infections which have been phagocytosed by a macrophage,whereby, it becomes possible to treat infections due to thesemicrooraganisms. As described above, unlike 9K granulysin, cytotoxicityis not perceived in 15K granulysin per se, and effects as a remedy forinfectious diseases having little side effect due to administration areexpected.

(ii) Intracorporeal Expression Vector for 15K Granulysin.

An active ingredient of the present remedy in this form is a recombinantvector in which a gene encoding 15K granulysin used for expressing theaforementioned recombinant protein is inserted into an intracorporealexpression vector.

Examples of the intracorporeal expression vector are not limited to, butinclude an adenovirus vector and a retrovirus vector.

For a recombinant intracorporeal expression vector, for example, anexpressible gene in which 15K granulysin is further transduced into acosmid vector in which the aforementioned virus gene has beentransduced, is incorporated and then, this cosmid vector and a parentvirus DNA—TP which have been restriction enzyme treated are transfectedinto 293 cells, whereby, homologous recombination occurs in the 293cells, thus, a desired intracorporeal expression vector is produced.

B. Form of Present Remedy

(i) Present Remedy Using Recombinant Protein of 15K Granulysin as ActiveIngredient.

In the first form of the present remedy, 15K granulysin is incorporatedas an active ingredient and, at the same time, an appropriatepharmaceutical preparation carrier can be incorporated to prepare into aform of a preparation composition (of course, only 15K glanulysin ispossible). As a pharmaceutical preparation carrier, for example,excipients and diluents such as fillers, bulking agents, binders,wetting agents, stabilizers, solubilizers, disintegrating agents andsurface active agents which can be conventionally used as apharmaceutical preparation carrier can be appropriately selected freelydepending on a specific dosage form. A form of a preparation compositionis not particularly limited as far as it is such a form that 15Kgranulysin can be effectively used in utilities of treatment ofinfectious diseases. For example, even solid agents such as tablets,powders, granules and pills may be formulated into an injectable formsuch as solutions, suspensions and emulsions. By adding an appropriatecarrier to 15K granulysin, a dried agent can be obtained which can bemade to be liquid upon use.

A dose of the thus obtained present remedy can be appropriately selecteddepending on an administration method and an administration form of anagent, and symptom of a patient, being not particularly limited.

Such the various forms of pharmaceutical preparations can beadministered by an appropriate administration route depending on itsform, for example, by intravenous, intramuscular, intraosseous,intra-articular, subcutaneous, intracutaneous or intraperitonealadministration in the case of an injectable form, or by oral or enteraladministration in the case of a solid agent form.

(ii) Present Remedy Using Intracorporeal Expression Vector for 15KGranulysin as an Active Ingredient.

By isolating and purifying the intracorporeal expression vector whichcan be prepared as described above, and administering this to a livingbody, 15K granulysin is produced in a living body, and thepharmacological effect of this 15K granulysin can be exerted.

A dosage form in this case is generally an injectable form, and theremedy can be administered by intravenous, intramuscular, intraosseous,intra-articular, subcutaneous, intracutaneous or intraperitonealadministration. A dose of such the present remedy can be appropriatelyselected depending on an administration method and an administrationform of an agent, and symptom of a patient, being not limited.Generally, it is preferable to appropriately prepare an intracorporealexpression vector expressing 15K granulysin as an active ingredient, andadminister this preparation at an appropriate amount once per day or bydividing into a few times per day.

EXAMPLE

Examples of the present invention will be described below.

[Test Example] Study of Antibacterial effect on Mycobacteriumtuberculosis when 15K Granulysin and a Macrophage are Present Jointly

(1) Preparation of Monoclonal Antibody Specific for 15K Granulysin

A RNA was extracted from human peripheral blood lymphocyte which hadbeen cultured in the presence of 100 to 200 unit/ml of IL-2 (2×10₆cells/ml of human peripheral blood lymphocytes were cultured at 37° C.under 5% CO₂ incubator for 10 days in RPMI1640 medium containing 10%fetal bovine serum) by the conventional method, and a RT-PCR method (PCRprimer1: SEQ ID NO:2, PCR primer 2:SEQ ID NO: 3) was performed usingthis RNA as a template, to synthesize a gene part containing a regionencoding a full length protein of 15K granulysin (Jongstra et al., J.Exp. Med, 165, 601: part corresponding to amino acid sequence of SEQ IDNO:1] as the amplification product of a complementary DNA (cDNA). ThiscDNA encoding a full length protein of 15K granulysin was incorporatedinto pRc/CMV or pcDL.SRa296 which is a mammal expression vector, theresulting recombinant vector was dissolved in a physiological saline,and a mouse was immunized with the solution subcutaneously orintracutaneously. After 4 to 5 times immunization at an interval of 1 to2 weeks, spleen cells obtained from mouse for which increase in anantibody titer was perceived by an indirect fluorescent antibody method(performed according to the method described later) was cell-fusedaccording to the conventional method, and a hybridoma producing anantibody which specifically binds to granulysin was retrieved again withan indirect fluorescent antibody method. That is, the cells weretransfected with the aforementioned gene encoding 15K granulysin, cells(Cos7) in which the gene was expressed was fixed with 4%paraformaldehyde, membranes were solubilized with 0.5% Tween 20, theculture supernatant of hybridoma cells was added thereto to react withan antibody, this was reacted with a fluorescently labeled anti-mouseIgG antibody, and fluorescence was detected, whereby, a hybridomaproducing an antibody which specifically binds to granulysin wasscreened. As a result, 9 hybridcmas producing an antibody whichspecifically binds to granulysin were obtained. Using the culturesupernatant of each of the resulting hybridomas, ammonium sulfateprecipitation and purification by Protein G column were performed toprepare two kinds of monoclonal antibodies to 15K granulysin.Hereinafter, this is referred to monoclonal antibody RF10 (which bindsto 15K granulysin, but does not bind to 9K granulysin) or monoclonalantibody RC8 (which binds to both 15K granulysin and 9K granulysin).

(2) Preparation of polyclonal antibody to 15K granulysin. A rabbit wasimmunized with a conjugate of a partial amino acid sequence (29 aminoacids) of granulysin (J. Exp. Med. 165:601-614 (1987), J. Exp. Med.,172:1159-1163 (1990)): Arg Thr Gly Arg Ser Arg Trp Arg Asp Val Cys ArgAso Phe Met Arg Arg Tyr Gln Ser Arg Val Thr Gln Gly Leu Val Ala Gly(N5-1: SEQ ID NO: 4) and limpet hemocyanin according to the conventionalmethod, to obtain anti-serum. The resulting antiserum was purified byammonium sulfate precipitation and Protein G column, and furtherpurified by affinity chromatography using a column bound with theaforementioned synthetic peptide (N5-1), to prepare a polyclonalantibody (anti-N5-1 antibody) to granulysin.

(3) Preparation of Granulysin Containing Culture Supernatant.

A gene recombinant vector was produced, in which, among a nucleotidesequence of SEQ ID NO:1, a nucleotide chain of a nucleotide sequenceencoding a part corresponding to 15K granulysin protein was incorporatedinto pFLAG-CMV vector (manufactured by Sigma) according to theconventional method. As a control, the pFLAG-CMV vector in whichrecombination with a gene was not performed, was used. Each of thesegene recombinant vectors was transfected into a Cos7 cell, and this wascultured at 37° C. under 5% CO₂ for 72 hours in DMEM medium(manufactured by Gibco). After completion of culture, the culture wascentrifuged (2500 rpm·20 min·4° C.) to obtain the culture supernatant.

Regarding each of the culture supernatants, the proteins were separatedby SDS-PAGE. After the protein was transferred to a nylon membrane froma gel of electrophoresed SDS-PAGE, the membrane was blocked withblocking solution (1% skim milk/washing solution), to this was boundmonoclonal antibody RF10 specific for 15K granulysin, and enzyme-labeledanti-mouse antibody and color developing substrate were acted on these,to develop a bond. As a result, in the supernatant obtained byexpressing a gene encoding 15K granulysin protein, a band exhibiting amolecular weight of 15K appeared. However, in the control, this bond wasnot perceived In addition, when the similar test was performed using apolyclonal antibody N5-1 binding to 15K and 9K granulysins, a bandshowing 15K was perceived, but a band showing 9K was not perceived.

From this result, it was revealed that 15K granulysin was specificallypresent in the aforementioned granulysin culture supernatant, butgranulysin was not present in control culture supernatant.

(4) Study of Antibacterial Effect

Lymphocyte separated from human blood was suspended in a culture(RPMI1640, 10% human serum) to around 2×107 cells in a plastic cultureplate (24 wells/plate) per 1 ml of medium according to the conventionalmethod, each 1 ml of this was dispensed into each well, allowed to standat 37° C. for 24 hours, lymphocyte was adhered to a plate surface walland, in this adherent macrophage, antibacterial effect of 15K granulysinwas studied.

Then, 1 ml of RFMI1640 (10% human serum) was added to a well,Mycobacterium tuberculosis (M. tuberculosis) (H37Rv, 1×10⁵ to 1×10⁶cfu)was static-cultured at 37° C. for 4 to 12 hours under 5% CO₂, to infectmacrophages with M. tuberculosis. After completion of infection, each 1ml of supernatant containing 15K granulysin (Grn supernatant) andsupernatant in the absence of 15K granulysin (Cont supernatant) wasadded to a well, and static culture was performed 2 to 12 hours underthe same conditions.

After completion of culture, the culturing supernatant was removed, theadhered cells in a plate were washed with PBS three times, M.tuberculosis in a cell was extracted with total 5 ml of 1% aqueoussaponin solution, this extract was diluted, seeded on a plate agarmedium [7H1 1 medium (manufactured by Gibco)], this was static-culturedat 37° C. for 14 days, and the number of colonies was counted.

The results are shown in FIG. 1.

In FIG. 1, Cont denotes control culturing supernatant, and Grn denotesgranulysin culture supernatant. A coordinate axis denotes the number ofcolonies. 1 on an abscissa axis denotes the results obtained bycontacting macrophages, M. tuberculosis and the culture supernatant, asdescribed above. 2 denotes the result obtained by static-culturing eachculture supernatant and M. tuberculosis in the absence of macrophages asin 1, washing this, static-culturing this in a 7H11 plate agar medium,and influence of M. tuberculosis on non-specific adsorption in a platewas confirmed. 3 denotes the result obtained by static culture each 1 mlof the culture supernatant and M. tuberculosis at 37° C. for 2 hours inthe aforementioned culture plate, and static-culture this in a 7H11plate agar medium.

As a result, it was revealed that 15K granulysin has antibacterialeffect on M. tuberculosis by intervention of macrophages. Whenmacrophages did not intervene, antibacterial effect of 15K granulysin onM. tuberculosis was not perceived.

By the aforementioned Test Example, antibacterial activity was perceivedin 15K granulysin, and it was revealed that 15K granulysin can be usedas an active ingredient of a remedy for infections.

It has been reported that, when 15K granulysin was transfected intocells such as Cos7 cells, HeLa and PC12, 15K granulysin was detected inthe culture supernatant, but damage was not perceived in the cells. When9K granulysin was used in place of 15K granulysin in the presence ofperforin, lo cytotoxic activity or apoptosis is induced (Pena, S. V. etal, J. Immunol., 158, 2680-2688(1997)).

1. A remedy for infectious diseases, which comprises 15K granulysin asan active ingredient.
 2. The remedy for infectious diseases according toclaim 1, wherein 15K granulysin is a recombinant protein.
 3. A remedyfor infectious diseases, which comprises, as an active ingredient, anintracorporeal expression vector for 15K granulysin, in which a geneencoding 15K granulysin is incorporated